Modified tgf-beta2 oligonucleotides

ABSTRACT

The invention refers to an oligonucleotide consisting of 10 to 18 nucleotides of selected regions of the TGF-beta2 nucleic acid sequence, which comprises modified nucleotides such as LNA, ENA, polyalkylene oxide-, 2′-fluoro, 2′-O-methoxy and/or 2′-O-methyl modified nucleotides. The invention further relates to pharmaceutical compositions comprising such oligonucleotide, wherein the composition or the oligonucleotide is used in the prevention and/or treatment of a malignant and/or benign tumor, an immunologic disease, fibrosis, or an ophthalmic disease such as dry eye, glaucoma or posterior capsular opacification (PCO).

CROSS REFERENCE TO RELATED APPLICATIONS

The invention is a divisional and claims priority to U.S. applicationSer. No. 14/779,945, filed on Sep. 24, 2015, which is a national phaseof PCT/EP2014/056232, filed on Mar. 27, 2014, which claims the benefitof priority to European Patent Application No. 13199838.7, filed Dec.30, 2013, European Patent Application No. 13173078.0, filed Jun. 20,2013, and European Patent Application No. 13161474.5, filed Mar. 27,2013, the entire contents of each of which are hereby incorporated intotal by reference.

SEQUENCE LISTING

This application incorporates by reference the Sequence Listingcontained in an ASCII text file named “362346_00047_SeqList.txt”submitted via EFS-Web. The text file was created on Dec. 8, 2017, and is12 kb in size.

BACKGROUND OF THE INVENTION

The invention is directed to oligonucleotides consisting of 10 to 18nucleotides hybridizing with the TGF-beta2 nucleic acid sequence, theTGF-beta1 or TGF-beta3 nucleic acid sequence, wherein theoligonucleotide comprises a modified nucleotide such as LNA, ENA,polyalkylene oxide-, 2′-fluoro, 2′-O-methoxy and/or 2′-O-methyl modifiednucleotides.

Transforming growth factor beta (TGF-beta) is a protein that controlsproliferation, cellular differentiation, and other functions in mostcells. It is a type of cytokine which plays amongst others a role inimmunity, cancer, heart disease, diabetes, Marfan syndrome, Loeys-Dietzsyndrome, Parkinson's disease, and AIDS.

TGF-beta is a secreted protein that exists in at least three isoforms(TGF-beta1. TGF-beta2 and TGF-beta3) encoded by different genes butsharing strong sequence and structure homologies. TGF-beta acts as anantiproliferative factor in normal epithelial cells and at early stagesof oncogenesis. However, later in tumor development TGF-beta can becometumor promoting through mechanisms including the induction ofepithelial-to-mesenchymal transition (EMT), a process that is thought tocontribute to tumor progression, invasion and metastasis (see“Glycoproteomic analysis of two mouse mammary cell lines duringtransforming growth factor (TGF)-beta induced epithelial to mesenchymaltransition” 7^(th) space.com.2009-01-08. Retrieved: 2009 Jan. 29).

In normal (epithelial) cells, TGF-beta stops the cell cycle at the G1stage (and stops cell proliferation), induce differentiation, or promoteapoptosis. When a cell is transformed into a cancer cell, TGF-beta nolonger suppresses cell proliferation, which is often the result ofmutations in the signaling pathway, and cancer cells proliferate.Proliferation of stromal fibroblasts is also induced by TGF-beta. Bothcells increase their production of TGF-beta. This TGF-beta acts on thesurrounding stromal cells, immune cells, endothelial, smooth-musclecells, and tumor microenvironment (see Pickupet al., “The roles of TGFβin the tumour microenvironment”. Nature Reviews Cancer (2013), 13:788-799). Thereby, it promotes angiogenesis, and by suppressingproliferation and activation of immune cells it causesimmunosuppression.

TGF-beta1-deficient mice die from cardiac, pulmonary, and gastricinflammation, suggesting that TGF-beta has a vital role in suppressingthe activation and proliferation of inflammatory cells. Smad3 is one ofthe key elements in TGF-beta dependent downstream signaling pathways.Smad3-deficient mice develop chronic mucosal infections due toimpairment of T-cell activation and mucosal immunity, suggesting a keyrole for TGF-beta in these processes. With respect to cancer, theproduction and secretion of TGF-beta by certain cancer cells suppressthe activities of infiltrating immune cells, thereby helping the tumorescape host immunosurveillance. This immunosuppressive effect may beanother important mechanism by which TGF-beta stimulates the growth oflate-stage tumors (see Blobe G C et al., May 2000. “Role of transforminggrowth factor beta in human disease”. N. Engl. J. Med. 342 (18),1350-1358). TGF-beta also converts effector T-cells, which normallyattack cancer with an inflammatory (immune) reaction, into regulatory(suppressor) T-cells, which turn off the inflammatory reaction.

Further, TGF-beta is one of the most potent regulators of the productionand deposition of extracellular matrix. It stimulates the production andaffects the adhesive properties of the extracellular matrix by two majormechanisms. First, TGF-beta stimulates fibroblasts and other cells toproduce extracellular-matrix proteins and cell-adhesion proteins,including collagen, fibronectin, and integrins. Second, TGF-betadecreases the production of enzymes that degrade the extracellularmatrix, including collagenase, heparinase, and stromelysin, andincreases the production of proteins that inhibit enzymes that degradethe extracellular matrix, including plasminogen-activator inhibitor typeI and tissue inhibitor of metalloprotease. The net effect of thesechanges is to increase the production of extracellular-matrix proteinsand either to increase or to decrease the adhesive properties of cellsin a cell-specific manner. In many cancer cells the production ofTGF-beta is increased, which increases the invasiveness of the cells byincreasing their proteolytic activity and promoting their binding tocell-adhesion molecules (see Blobe G C et al., May 2000, “Role oftransforming growth factor beta in human disease”, N. Engl. J. Med. 342(18), 1350-1358).

Thus, therapeutic agents which are able to influence TGF-beta expressionand activity, respectively, are essential in particular for use inpreventing and/or treating TGF-beta linked diseases. EP 1008649 and EP0695354, for example, disclose oligonucleotides hybridizing with themRNA of TGF-beta1 and/or TGF-beta2, and which are suitable to be usedfor manufacturing pharmaceutical compositions for example for preventingand/or treating cancer. None of these oligonucleotides comprisesmodifications such as LNA, ENA etc.

WO 2003/85110, WO 2005/061710, and WO 2008/1138904 for example refer tooligonucleotides comprising modifications of the nucleotides, which aredirected to the inhibition of HIF-1A, Bcl-2 and HER3, respectively, andusable in the treatment of cancer.

Criteria for the selection of oligonucleotides are mainly the length ofthe oligonucleotide, the GC-percentage, the tendency for hairpinformation, dimerization and the melting temperature (Tm). In general,high Tm (melting temperature) is preferred. Furthermore, theoligonucleotides must be specific for the target mRNA and shall nothybridize to non-target mRNAs in order to decrease potential off-targeteffects.

Hence, there is a high scientific and medical need for therapeuticagents, which reduce or inhibit TGF-beta expression and/or activity.Particularly, there is a long-standing need for oligonucleotides such asantisense oligonucleotides, which specifically interact and thus, reduceor inhibit the expression of TGF-beta1, TGF-beta2, and/or TGF-beta3, aswell as oligonucleotides, which specifically inhibit TGF-beta1 andTGF-beta2, or TGF-beta1 and TGF-beta3, or TGF-beta2 and TGF-beta3,without causing any (severe) side effects.

SUMMARY OF THE INVENTION

The present invention refers to oligonucleotides consisting of 10 to 18nucleotides of the TGF-beta2 nucleic acid sequence of SEQ ID NO. 1 (seeFIG. 1a and FIG. 1b ) wherein one or more nucleotide(s) of theoligonucleotide is/are modified. Preferred oligonucleotides comprisingor consisting of one of SEQ ID NO. 2 to 20 are presented in Table 1.These oligonucleotides are highly effective in the reduction andinhibition of TGF-beta2 expression and activity, respectively.

Preferred oligonucleotides of the present invention are ASPH47, ASPH190,ASPH191, ASPH192, ASPH193, ASPH194, ASPH195, ASPH196, ASPH197, ASPH198,ASPH199, ASPH200. ASPH201, and ASPH202, ASPH203, ASPH204, ASPH205,ASPH206, ASPH207, ASPH208, ASPH209, ASPH210, ASPH211, ASPH212, ASPH213,ASPH214, ASPH215, ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221,ASPH222, and ASPH223 respectively.

Modifications of one or more nucleotides of the oligonucleotides of thepresent invention are selected from the group consisting of LNA, ENA,polyalkylene oxide such as triethylene glycol (TEG), 2′-fluoro,2′-O-methoxy and 2′-O-methyl. The modifications are preferably locatedat the 5′- and/or 3′-end of the oligonucleotide. An oligonucleotidecomprising such modified nucleotide is a modified oligonucleotide.

Modified nucleotides are for example arranged in a row, one directlynext to the other, or in different patterns, where one or moreunmodified nucleotides follow a modified nucleotide. For example anoligonucleotide starts with one or more modified nucleotides followed byone or more, e.g., one, two, three or four, unmodified or unlockednucleotides followed again by one or more modified nucleotides. In oneembodiment both ends of the oligonucleotide comprise an identicalpattern of modified and unmodified or unlocked nucleotides. In anotherembodiment, the pattern of modifications at the 3′- and 5′-end differincluding that one end does not comprise a modified nucleotide.Preferably the modified oligonucleotides comprise a series of 8 or 9unlocked nucleotides.

Alternatively, a nucleotide at any other position in the oligonucleotideis modified, or at least one nucleotide at the 5′- and/or 3′-end of theoligonucleotide and at any other position in the oligonucleotide. Theoligonucleotides comprise either one type of modification, or one ormore different modifications. Optionally, at least one phosphate linkagebetween two consecutive nucleotides (modified or unmodified) of theoligonucleotide is a phosphorothioate or a methylphosphonate. In apreferred embodiment, the oligonucleotides of the present invention arephosphorothioates.

All the oligonucleotides of the different embodiments are for use in amethod of the prevention and/or treatment of a malignant or a benigntumor, an immunologic disease, fibrosis (e.g., idiopathic pulmonaryfibrosis, renal fibrosis, kidney fibrosis), cirrhosis (e.g., livercirrhosis), scleroderma or related dermatologic diseases, or an eyedisease such as glaucoma or posterior capsular opacification (PCO), aCNS disease, hair loss etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a and FIG. 1b shows the nucleic acid sequence of the humanTGF-beta2 mRNA (NM_003238.3).

FIG. 2 presents examples of nucleotide modifications.

FIG. 3a (i), FIG. 3a (ii), FIG. 3a (iii), FIG. 3b (i), FIG. 3b (ii), andFIG. 3b (iii) shows the inhibition of the expression of TGF-beta1,TGF-beta2 and TGF-beta3 mRNA in human Panc-1 pancreatic cancer cells andmouse RenCa renal cell carcinoma cells. Pane-1 cells and RenCa cellswere treated with different modified oligonucleotides at a dose of 1.1μM in the absence of any transfection reagent (gymnotic transfection orunassisted transfection or gymnotic delivery), and inhibition of theTGF-beta1 (black columns), TGF-beta2 (white columns), and TGF-beta3(striped columns) mRNA expression was measured after 72 h. The figuresrefer to the results for the modified oligonucleotides ASPH190, ASPH191.ASPH192, ASPH193, ASPH194, ASPH195, ASPH196, ASPH197, ASPH198, ASPH199,ASPH200, ASPH201, ASPH202, ASPH203, ASPH204, ASPH205, ASPH206, ASPH207,ASPH208, ASPH209, ASPH210, ASPH211, ASPH212, ASPH213, ASPH214, ASPH215,ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221, ASPH222, andASPH223, respectively. The Figures present the inhibitory effect ofthese TGF-beta oligonucleotides in Panc-1 cells and in RenCa cells.

FIG. 4a and FIG. 4b depicts the inhibiting effect of oligonucleotides ofthe present invention on the expression of TGF-beta1 and TGF-beta2protein. Human Pane-1 cells were transfected with 20, 6.67, 2.22, 0.74,0.25, 0.08 or 0.009 μM of the modified oligonucleotide ASPH47 (FIG. 4a). Negative control is the scrambled oligonucleotide (scrLNA) of SEQ IDNo. 22 (FIG. 4b ) in concentrations of 40, 13.33, 4.44, 1.48, 0.49,0.16, 0.05, or 0.02 μM. TGF-beta1 (diamonds) and TGF-beta2 (squares)protein levels in cell supernatants were determined by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to oligonucleotides, in particularantisense oligonucleotides, which comprise at least one modifiednucleotide and are suitable to interact with TGF-beta mRNA, preferablywith TGF-beta1, TGF-beta2, and/or TGF-beta3. The oligonucleotidescomprise or consist of 10 to 18, nucleotides of the TGF-beta2 nucleicacid according to SEQ ID NO. 1. Most preferred the oligonucleotidecomprises or consists of 10, 11, 12, 13, 14, 15, 16, 17, or 18nucleotides. The oligonucleotide is a single or double stranded RNA orDNA, including siRNA, microRNA, apatmer or spiegelmer. Preferably, theoligonucleotide is an antisense oligonucleotide.

Preferred oligonucleotides of the present invention are ASPH47, ASPH90,ASPH191, ASPH192, ASPH193, ASPH194, ASPH195, ASPH1196, ASPH197, ASPH198,ASPH199, ASPH200, ASPH201, and ASPH202, ASPH203, ASPH204, ASPH205,ASPH206, ASPH207. ASPH208, ASPH209, ASPH210, ASPH211, ASPH212, ASPH213,ASPH214, ASPH215, ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221,ASPH222, and ASPH223 respectively. The antisense oligonucleotides of thepresent invention can be described differently, e.g., ASPH47, ASPH0047,ASPH_47 or ASPH_0047 referring to the same oligonucleotide.

A nucleotide forms the building block of an oligonucleotide, and is forexample composed of a nucleobase (nitrogenous base, e.g., purine orpyrimidine), a five-carbon sugar (e.g., ribose, 2-deoxyribose,arabinose, xylose, lyxose, allose, altorse, glucose, mannose, gulose,idose, galactose, talose or stabilized modifications of those sugars),and one or more phosphate groups. Examples of modified phosphate groupsare phosphorothioate or methylphosphonate. Each compound of thenucleotide is modifiable, and is naturally or non-naturally occurring.The latter are for example locked nucleic acid (LNA), a2′-O,4′-C-ethylene-bridged nucleic acid (ENA), polyalkylene oxide- (suchas triethylene glycol (TEG)), 2′-fluoro, 2′-O-methoxy and 2′-O-methylmodified nucleotides as described for example by Freier & Altmann (Nucl.Acid Res., 1997, 25, 4429-4443) and Uhlmann (Curr. Opinion in Drug &Development (2000, 3 (2): 293-213), which are shown in FIG. 2.

A LNA is a modified RNA nucleotide, wherein the ribose moiety ismodified with an extra bridge connecting the 2′ oxygen and 4′ carbon(2′-4′ribonucleoside). The bridge “locks” the ribose in the 3′-endo(North) conformation, which is often found in the A-form duplexes. LNAnucleosides and nucleotides, respectively, comprise for example theforms of thio-LNA, oxy-LNA, or amino-LNA, in alpha-D- orbeta-L-configuration, and are mixable and combinable, respectively, withDNA or RNA residues in the oligonucleotide.

The oligonucleotides of the present invention, i.e., modifiedoligonucleotides, comprise at least one modified nucleotide, preferablyLNA and/or ENA, at the 5′- and/or 3′-end of the oligonucleotide. In apreferred embodiment, the oligonucleotide comprises 1, 2, 3, or 4 LNAsor ENAs at the 5′-end, and 1, 2, 3, or 4 LNAs or ENAs at the 3′-end. Inanother preferred embodiment, the oligonucleotide comprises 1, 2, 3, or4 LNAs or ENAs at the 5′-end or 3′-end, and a polyalkylene oxide such asTEG at the 3′- or 5′-end. The modified oligonucleotides show asignificantly increased inhibition on TGF-beta expression and activity,respectively, which results in an improved prevention and/or treatmentof a malignant or benign tumor, an immunologic disease, fibrosis, eyedisease such as dry eye, glaucoma or posterior capsular opacification(PCO), CNS disease hair loss etc. The oligonucleotides of the presentinvention target TGF-beta linked diseases either by hybridization withTGF-beta mRNA, preferably TGF-beta1, TGF-beta2, or TGF-beta3.

Preferably two or more oligonucleotides are combined, wherein at leastone oligonucleotide specifically inhibits TGF-beta1 and at least oneoligonucleotide specifically inhibits TGF-beta2, or wherein at least oneoligonucleotide specifically inhibits TGF-beta1 and at least oneoligonucleotide specifically inhibits TGF-beta3, or wherein at least oneoligonucleotide specifically inhibits TGF-beta2 and at least oneoligonucleotide specifically inhibits TGF-beta3, or wherein at least oneoligonucleotide specifically inhibits TGF-beta1, at least oneoligonucleotide specifically inhibits TGF-beta2, and at least oneoligonucleotide specifically inhibits TGF-beta3. The oligonucleotide ofthe present invention most preferably inhibits the expression and/oractivity of TGF-beta2 mRNA.

In another embodiment, one oligonucleotide inhibits two TGF-betaisoforms such as TGF-beta1 and TGF-beta2, TGF-beta2 and TGF-beta3, orTGF-beta1 and TGF-beta3. An oligonucleotide inhibiting the expression oftwo or all three isoforms—TGF-beta1, TGF-beta2, and TGF-beta3—is definedas pan-specific oligonucleotide.

In a further embodiment three or more oligonucleotides are combined,wherein at least one oligonucleotide specifically inhibits TGF-beta1,another oligonucleotide specifically inhibits TGF-beta2, and a furtheroligonucleotide specifically inhibits TGF-beta3, and optionally one ormore additional oligonucleotides inhibiting TGF-beta1, TGF-beta2 orTGF-beta3.

The oligonucleotides of the present invention have for example an IC₅₀in the range of 0.1 to 20 μM, preferably in the range of 0.2 to 15 μM,more preferably in the range of 0.4 to 10 μM, and even more preferred inthe range of 0.5 to 5 μM.

The present invention further refers to a pharmaceutical compositioncomprising an oligonucleotide according to the invention as activeingredient. The pharmaceutical composition comprises at least oneoligonucleotide of the present invention and optionally further anantisense compound, an antibody, a chemotherapeutic compound, ananti-inflammatory compound, an antiviral compound and/or animmuno-modulating compound. Pharmaceutically acceptable binding agentsand adjuvants are optionally comprised by the pharmaceuticalcomposition.

In one embodiment, the oligonucleotide and the pharmaceuticalcomposition, respectively, is formulated as dosage unit in form of asolution comprising binders, excipients, stabilizers etc.

The oligonucleotide and/or the pharmaceutical composition isadministrable via different routes. These routes of administrationinclude, but are not limited to, electroporation, epidermal, impressioninto skin, intra-arterial, intra-articular, intracranial, intradermal,intra-lesional, intra-muscular, intranasal, intra-ocular, intrathecal,intracameral, intraperitoneal, intraprostatic, intrapulmonary,intraspinal, intratracheal, intratumoral, intravenous, intravesical,placement within cavities of the body, nasal inhalation, oral, pulmonaryinhalation (e.g., by inhalation or insufflation of powders or aerosols,including by nebulizer), subcutaneous, subdermal, topical (includingophthalmic and to mucous membranes including vaginal and rectaldelivery), or transdermal.

For parenteral, subcutaneous, intradermal or topical administration theoligonucleotide and/or the pharmaceutical composition include forexample a sterile diluent, buffers, regulators of toxicity andantibacterials. In a preferred embodiment, the oligonucleotide orpharmaceutical composition is prepared with carriers that protectagainst degradation or immediate elimination from the body, includingimplants or microcapsules with controlled release properties. Forintravenous administration the preferred carriers are for examplephysiological saline or phosphate buffered saline. An oligonucleotideand/or a pharmaceutical composition comprising such oligonucleotide fororal administration includes for example powder or granule,microparticulate, nanoparticulate, suspension or solution in water ornon-aqueous media, capsule, gel capsule, sachet, tablet or minitablet.An oligonucleotide and/or a pharmaceutical composition comprising forparenteral, intrathecal, intracameral or intraventricular administrationincludes for example sterile aqueous solutions which optionally containbuffer, diluent and other suitable additive such as penetrationenhancer, carrier compound and other pharmaceutically acceptable carrieror excipient.

A pharmaceutically acceptable carrier is for example liquid or solid,and is selected with the planned manner of administration in mind so asto provide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include, butare not limited to, a binding agent (e.g. pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); filler(e.g. lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricant (e.g., magnesium stearate, talcum,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrate (e.g., starch, sodiumstarch glycolate, etc.); or wetting agent (e.g., sodium lauryl sulphate,etc.). Sustained release oral delivery systems and/or enteric coatingsfor orally administered dosage forms are described in U.S. Pat. Nos.4,704,295; 4,556,552; 4,309,406; and 4,309,404. An adjuvant is includedunder these phrases.

Beside being used in a method of human disease prevention and/ortreatment, the oligonucleotide and/or the pharmaceutical compositionaccording to the present invention is also used in a method forprevention and/or treatment of other subjects including veterinaryanimals, reptiles, birds, exotic animals and farm animals, includingmammals, rodents, and the like. Mammals include for example horses,dogs, pigs, cats, or primates (for example, a monkey, a chimpanzee, or alemur). Rodents include for example rats, rabbits, mice, squirrels, orguinea pigs.

The oligonucleotide or the pharmaceutical composition according to theinvention is used in a method for the prevention and/or treatment ofmany different diseases, preferably benign or malignant tumors,immunologic diseases, bronchial asthma, heart disease, fibrosis (e.g.,liver fibrosis, idiopathic pulmonary fibrosis, liver cirrhosis, kidneycirrhosis, scleroderma), diabetes, wound healing, disorders of theconnective tissue (e.g., in heart, blood vessel, bone, joint, eye suchas the Marfan or Loeys-Dietz syndrome), psoriasis, eye diseases (e.g.,glaucoma, posterior capsular opacification (PCO), retinoblastoma,choroidcarcinoma, macular degeneration, such as age-related maculardegeneration, diabetic macular endma, or cataract), CNS disease (e.g.,Alzheimer's disease, Parkinson's disease), coronary atherosclerosis(coronary intervention or coronary artery bypass graft (CABG) surgery orhair loss. A tumor is for example selected from the group of solidtumors, blood born tumors, leukemias, tumor metastasis, hemangiomas,acoustic neuromas, neurofibromas, trachomas, pyogenic granulomas,astrocytoma such as anaplastic astrocytoma, acoustic neuroma, blastoma,Ewing's tumor, craniopharyngloma, ependymoma, medulloblastoma, glioma,glioblastoma, hemangloblastoma, Hodgkins-lymphoma, medullablastoma,leukaemia, melanoma such as primary and/or metastatic melanoma,mesothelioma, mycloma, neuroblastoma, neurofibroma, non-Hodgkinslymphoma, pinealoma, retinoblastoma, sarcoma, seminoma, trachomas,Wilm's tumor, bile duct carcinoma, bladder carcinoma, brain tumor,breast cancer, bronchogenic carcinoma, carcinoma of the kidney, cervicalcancer, choriocarcinoma, cystadenocarcinome, embryonal carcinoma,epithelial carcinoma, esophageal cancer, cervical carcinoma, coloncarcinoma, colorectal carcinoma, endometrial cancer, gallbladder cancer,gastric cancer, head cancer, liver carcinoma, lung carcinoma, medullarycarcinoma, neck cancer, non-small-cell bronchogenic/lung carcinoma,ovarian cancer, pancreas carcinoma, papillary carcinoma, papillaryadenocarcinoma, prostate cancer, small intestine carcinoma, prostatecarcinoma, rectal cancer, renal cell carcinoma (RCC, e.g., clear cellRCC, papillary RCC, chromophobe RCC), oncocytoma kidney cancer,transitional cell kidney cancer, skin cancer, small-cellbronchogenic/lung carcinoma, squamous cell carcinoma, sebaceous glandcarcinoma, testicular carcinoma, and uterine cancer. The oligonucleotideor the pharmaceutical composition of the present invention is not onlyused in a method for the prevention and/or treatment of a tumor, butlikewise on a metastasis.

The present invention is preferably directed to an oligonucleotide foruse in a method for prevention and/or treatment of ophthalmic diseasessuch as, but not limited to, retinoblastoma, choroidcarcinoma, glaucoma,posterior capsular opacification, dry eye, macular degeneration. e.g.,age-related macular degeneration, diabetic macular endma, cataract,proliferative vitreoretinopathy, Marfan or Loeys-Dietz syndrome.

The antisense oligonucleotides of the present invention arecharacterized in that they show an unexpected low toxicity and thus, arewell tolerated by different organisms. They oligonucleotides show areasonable distribution in the organism, wherein highest concentrationsare measured in the kidney, liver, skin and spleen.

The present invention provides numerous oligonucleotides, which arehighly efficient in the reduction and inhibition, respectively, ofTGF-beta, in particular TGF-beta2 mRNA expression due to the specificselection of the sequence of the oligonucleotide and the modification ofthe nucleotide. The following Table 1 shows numerous preferred modifiedoligonucleotides according to the present invention (modifiednucleosides are indicated in bold letters). Each oligonucleotide isdefined as ASPH and a number, which is defined by a specific sequenceand modification of the nucleosides:

SEQ ID NO. Sequence Modification ASPH  2 CAAAGTATTTGGTCT LNA 4 + 447 or 193 CC  3 AGTATTTGGTCTCC LNA 3 + 3 190 or M12- ASPH47  4AAGTATTTGGTCTC LNA 3 + 3 191 or M9- ASPH47  5 AAGTATTTGGTCTCC LNA 3 + 3192 or M8- ASPH47  6 AGTATTTGGTCTCC LNA 2 + 3 194  6 AGTATTTGGTCTCC1LNA + 1N + 1LNA + 8N + 3LNA 195  6 AGTATTTGGTCTCC 3LNA + 8N + 1LNA +1N + 1LNA 196  6 AGTATTTGGTCTCC LNA 3 + 2 197  6 AAGTATTTGGTCTC LNA 4 +2 198  7 AGTATTTGGTCTCCA 3LNA + 8N + 1LNA + 1N + 2LNA 199  7AGTATTTGGTCTCCA 3LNA + 8N + 2LNA + 1N + 1LNA 200  7 AGTATTTGGTCTCCA2LNA + 1N + 1LNA + 8N + 3LNA 201  7 AGTATTTGGTCTCCA 1LNA + 1N + 2LNA +8N + 3LNA 202  7 AGTATTTGGTCTCCA LNA 3 + 2 203  7 AGTATTTGGTCTCCALNA 2 + 3 204  7 AGTATTTGGTCTCCA LNA 2 + 4 205  8 AAGTATTTGGTCTCC 3LNA +8N + 1LNA + 1N + 2LNA 206  8 AAGTATTTGGTCTCC 3LNA + 8N + 2LNA + 1N +1LNA 207  8 AAGTATTTGGTCTCC 2LNA + 1N + 1LNA + 8N + 3LNA 208  8AAGTATTTGGTCTCC 1LNA + 1N + 2LNA + 8N + 3LNA 209  8 AAGTATTTGGTCTCCLNA 3 + 2 210  8 AAGTATTTGGTCTCC LNA 2 + 3 211  2 CAAAGTATTTGGTCTLNA 3 + 3 212 CC  2 CAAAGTATTTGGTCT LNA 2 + 2 213 CC  2 CAAAGTATTTGGTCT1LNA + 1N + 2LNA + 8N + 3LNA 214 CC  2 CAAAGTATTTGGTCT 1LNA + 3N +1LNA + 8N + 3LNA 215 CC  2 CAAAGTATTTGGTCT 1LNA + 2N + 2LNA + 8N + 4LNA216 CC  2 CAAAGTATTTGGTCT 1LNA + 2N + 2LNA + 8N + 1LNA + 217 CC 1N +2LNA  2 CAAAGTATTTGGTCT 1LNA + 1N + 3LNA + 8N + 3LNA 218 CC  2CAAAGTATTTGGTCT 1LNA + 1N + 2LNA + 8N + 3LNA 219 CC  2 CAAAGTATTTGGTCT1LNA + 2N + 3LNA + 8N + 2LNA 220 CC  2 CAAAGTATTTGGTCT 1LNA + 2N +3LNA + 8N + 1LNA + 221 CC 1N + 1LNA  2 CAAAGTATTTGGTCT LNA 3 + TEG 222CC-TEG  2 CAAAGTATTTGGTCT LNA 4 + TEG 223 CC-TEG  9 CAAAGTATTTGGTCTLNA 4 + 3 M1- C ASPH47 10 CAAAGTATTTGGTCT LNA 4 + 2 M2- ASPH47 11CAAAGTATTTGGTC LNA 4 + 1 M3- ASPH47 12 AAAGTATTTGGTCTC LNA 3 + 4 M4-ASPH47 13 AAAGTATTTGGTCTC LNA 3 + 3 M5- ASPH47 14 AAAGTATTTGGTCT LNA 3 +2 M6- ASPH47 15 AAAGTATTTGGTC LNA 3 + 1 M7- ASPH47 16 AAGTATTTGGTCTLNA 2 + 2 M10- ASPH47 17 AAGTATTTGGTC LNA 2 + 1 M11- ASPH47 18AGTATTTGGTCTC LNA 1 + 3 M13- ASPH47 19 AGTATTTGGTCT LNA 1 + 2 M14-ASPH47 20 AGTATTTGGTC LNA 1 + 1 M15- ASPH47

Table 1 shows the nucleic acid sequences of selected oligonucleotides ofthe present invention as well as the modifications of the nucleotides,wherein LNA 4+4 means 4×LNAs at the 5′- and 3′-end of theoligonucleotide are modified, wherein LNA 4+3 means 4×LNAs at the 5′-endand 3×LNAs at the 3′-end of the oligonucleotide are modified, whereinLNA 3-+4 means 3×LNAs at the 5′-end and 4×LNAs at the 3′-end of theoligonucleotide are modified, wherein LNA 3+3 means 3×LNAs at the 5′-and 3′-end of the oligonucleotide are modified, wherein LNA 3+2 means3×LNAs at the 5′-end and 2×LNAs at the 3′-end of the oligonucleotide aremodified, wherein LNA 2+3 means 2×LNAs at the 5′-end and 3×LNAs at the3′-end of the oligonucleotide are modified, wherein LNA 2+2 means 2×LNAsat the 5′- and 3′-end of the oligonucleotide are modified.Alternatively, some oligonucleotides comprise ENA 4+4, i.e., 4×ENA atthe 5′- and 3′-end of the oligonucleotide are modified, or ENA 3+3, i.e,3×ENA at the 5′- and 3′-end of the oligonucleotide are modified. Furtheroligonucleotides comprise 2′ O-meth 4+4, wherein the oligonucleotidecomprises 4×2′ O-methyl modified nucleotides at the 5′- and 3′-end ofthe oligonucleotide, or comprises 2′ fluoro 4+4, wherein theoligonucleotide comprises 4×2′ fluoro modified nucleotides at the 5′-and 3′-end. Oligonucleotides comprising LNA 3+TEG comprise 3×LNAs at the5′-end and one triethylene glycol (TEG) at the 3′-end of theoligonucleotide. Some oligonucleotides comprise LNAs which are notarranged in a row but are separated by an unlocked (unmodified)nucleoside having for example the sequences 1LNA+1N+1LNA+8N+3LNA,3LNA+8N+1LNA+1N+1LNA, 3LNA+8N+1LNA+1N+2LNA, 3LNA+8N+2LNA+1N+1LNA,2LNA+1N+1LNA+8N+3LNA, 1LNA+1N+2LNA+8N+3LNA, 1LNA+2N+2LNA+8N+3LNA,1LNA+3N+1LNA+8N+3LNA, 1LNA+2N+2LNA+8N+4LNA,1LNA+2N+2LNA+8N+1LNA+1N+2LNA, 1LNA 1N+3LNA+8N+3LNA,1LNA+1N+2LNA+8N+3LNA, 1LNA+2N+3LNA+8N+2LNA, or1LNA+2N+3LNA+8N+1LNA+1N+1LNA, wherein “N” is a nucleoside without lockedmodification. LNA nucleosides are indicated in the sequence in boldletters, and triethylene glycol is abbreviated as TEG in this table.“ASPH” in combination with a number refers to the differentoligonucleotides and their different modifications as described inTable 1. The antisense oligonucleotides of the present invention can bedescribed differently, e.g., ASPH47, ASPH0047, ASPH_47 or ASPH_0047referring to the same oligonucleotide. These modified oligonucleotideswere tested e.g. in experiments shown in the following examples.

For the purpose of clarity and a concise description, features aredescribed herein as part of the same or separate embodiments, however,it will be appreciated that the scope of the invention may includeembodiments having combinations of all or some of the featuresdescribed.

The following examples will serve to further illustrate the presentinvention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatthe scope of the present invention refers to various other embodiments,modifications, and equivalents thereof which, after reading thedescription herein, may suggest themselves to those skilled in the artwithout departing from the spirit of the invention.

EXAMPLES

In the following examples, the effect of the oligonucleotides listed inTable 1 has been tested in view of the reduction and inhibition,respectively, of TGF-beta1 and/or TGF-beta2 expression. SEQ ID NO. 21(T-LNA: CGGCATGTCTATTTTGTA, wherein 3× nucleotides at the 5′- and 3′-endare LNAs) and SEQ ID NO. 22 (scr-LNA: CGTTTAGGCTATGTACTT, wherein 3×nucleotides at the 5′- and 3′-end are LNAs) are used as controloligonucleotides, wherein SEQ ID NO. 22 (negative control) is thescrambled form of SEQ ID NO. 21 (positive control). The cells wereeither transfected in the presence of a transfecting agent (e.g.,Lipofectamine), or in the absence of any transfecting agent (which isdefined as gymnotic transfection or unassisted transfection or gymnoticdelivery). In case of gymnotic delivery, the entry of theoligonucleotide into the cell solely depends on the interaction of theoligonucleotide with the cell (no agent supports the entry). Therefore,gymnotic delivery is considered to reflect better conditions of the invivo settings.

Example 1

Either human Panc-1 pancreatic cancer cells (FIG. 3a (i), FIG. 3a (ii),and FIG. 3a (iii)) or mouse RenCa renal cell carcinoma cells (FIG. 3b(i), FIG. 3b (ii), and FIG. 3b (iii)) were treated with 1.1 μM ofASPH190, ASPH191, ASPH192, ASPH193, ASPH194, ASPH195, ASPH196, ASPH197,ASPH198, ASPH99, ASPH200, ASPH201, ASPH202, ASPH203, ASPH204, ASPH205,ASPH1206, ASPH207, ASPH208, ASPH209, ASPH210, ASPH211, ASPH212, ASPH213,ASPH214, ASPH215, ASPH216, ASPH217, ASPH218, ASPH219, ASPH220, ASPH221,ASPH222, or ASPH223 in the absence of a transfecting agent (gymnotictransfection or gymnotic delivery). The expression of TGF-beta1 (blackcolumn), TGF-beta2 (white column) and TGF-beta3 (striped column) mRNAwas determined 72 h after transfection. Significant reduction of theexpression of TGF-beta2 mRNA is demonstrated in FIG. 3a (i), FIG. 3a(ii). FIG. 3a (iii), FIG. 3b (i), FIG. 3b (ii), and FIG. 3b (iii). Thenegative control is scrambled LNA (scr LNA) of SEQ ID No. 22.

Example 2

Human Pane-1 pancreatic cancer cells were treated with 10 μM, 3.3 μM,1.1 μM, 0.37 μM, and 0.12 μM of ASPH47, M1-ASPH47, M2-ASPH47, M3-ASPH47,M4-ASPH47, M5-ASPH47, M6-ASPH47, M7-ASPH47, M8-ASPH47, M9-ASPH47,M10-ASPH47, M11-ASPH47. M12-ASPH47, M13-ASPH47, M14-ASPH47, orM15-ASPH47 in the absence of a transfecting agent (gymnotic transfectionor gymnotic delivery). The inhibitory effect of the modifiedoligonucleotides on expression of TGF-beta2 mRNA was determined 72 hafter treatment start. TGF-beta2 values were normalized to GAPDH andoligonucleotide concentrations resulting in 50% reduction of TGF-beta2mRNA (=IC₅₀ values) were calculated. Under gymnotic transfectionexperimental conditions, the oligonucleotides enter the cells andstrongly inhibit the expression of TGF-beta2 mRNA. The results of theexperiments are shown in Table 2:

oligos IC₅₀ (μM) M1_ASPH_0047 0.3 M2_ASPH_0047 0.49 M3_ASPH_0047 1.75M4_ASPH_0047 0.95 M5_ASPH_0047 0.85 M6_ASPH_0047 1.49 M7_ASPH_0047 n.a.M8_ASPH_0047 0.89 M9_ASPH_0047 1.05 M10_ASPH_0047 7.75 M11_ASPH_0047n.a. M12_ASPH_0047 1.58 M13_ASPH_0047 1.91 M14_ASPH_0047 n.a.M15_ASPH_0047 n.a. ASPH_0047 0.348All the modified oligonucleotides show an IC₅₀ in the submicromolar tolower submicromolar range, showing that they have extremely high potencyeven without the requirement of a transfection reagent.

Example 3

Human Pane-1 pancreatic cancer cells were transfected with 20, 6.67,2.22, 0.74, 0.25, 0.08 or 0.009 μM of the modified oligonucleotideASPH147, and results are shown in FIG. 4a . Negative control is thescrambled oligonucleotide (scr LNA) of SEQ ID No. 22 (FIG. 4b ). Cellswere transfected in the absence of a transfecting agent (gymnotictransfection or gymnotic delivery). The oligonucleotides were added tothe cells for 3 days, 37° C. TGF-beta1 and TGF-beta2 protein levels incell supernatants were determined by ELISA. ASPH47 specifically inhibitsthe expression of TGF-beta2 in a dose-dependent manner and does not showtarget inhibiting effect on TGF-beta1 (FIG. 4a ). The scrLNA of SEQ IDNo. 22 does not show any inhibiting effect on the expression ofTGF-beta1 or TGF-beta2, even if the concentrations were doubled (40,13.33, 4.44, 1.48, 0.49, 0.16, 0.05, or 0.02 μM) in comparison to theindividual concentrations of ASPH47. Results for TGF-beta1 are indicatedin diamonds, and results for TGF-beta2 in squares in FIGS. 4a and 4 b.

Embodiments

1. Antisense oligonucleotide consisting of 10 to 18 nucleotides of theTGF-beta2 nucleic acid sequence of SEQ ID NO. 1, wherein one or morenucleotide(s) of the oligonucleotide is/are modified, wherein themodified nucleotide is a LNA, and/or an ENA, polyalkylene oxide-,2′-fluoro-, 2′-O-methoxy-, and/or 2′O-methyl-modified nucleotide.2. Antisense oligonucleotide according to embodiment 1, wherein themodified nucleotide is located at the 5′- and/or 3′-end of theoligonucleotide.3. Antisense oligonucleotide according to embodiment 1 or 2, wherein theoligonucleotide consists of or comprises a sequence selected from thegroup consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO.10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ IDNO. 15, SEQ ID NO. 16, SEQ ID NO. 17, and SEQ ID NO. 18.4. Antisense oligonucleotide according to any one of embodiments 1 or 3,wherein the oligonucleotide is selected from the group consisting ofAGTATTTGGTCTCC (ASPH190), AAGTATTTGGTCTC (ASPH191), AAGTATTTGGTCTCC(ASPH192), CAAAGTATTTGGTCT (ASPH193), AGTATTTGGTCTCC (ASPH194),AGTATTTGGTCTCC (ASPH195), AGTATTTGGTCTCC (ASPH196), AGTATTTGGTCTCC(ASPH197), AAGTATTTGGTCTC (ASPH198), AGTATTTGGTCTCCA (ASPH199),AGTATTTGGTCTCCA (ASPH200), AGTATTTGGTCTCCA (ASPH201), AGTATTTGGTCTCCA(ASPH202), AGTATTTGGTCTCCA (ASPH203), AGTATTTGGTCTCCA (ASPH204),AGTATTTGGTCTCCA (ASPH205), AAGTATTTGGTCTCC (ASPH206), AAGTATTTGGTCTCC(ASPH207), AAGTATTTGGTCTCC (ASPH208), AAGTATTTGGTCTCC (ASPH209),AAGTATTTGGTCTCC (ASPH210), AAGTATTTGGTCTCC (ASPH211), CAAAGTATTTGGTCTCC(ASPH212), CAAAGTATTTGGTCTCC (ASPH213), CAAAGTATTTGGTCTCC (ASPH214),CAAAGCTATTTGGTCTCC (ASPH215), CAAAGTATTTGGTCTCC (ASPH216),CAAAGTATTTGGTCTCC (ASPH217), CAAAGTATTTGGTCTCC (ASPH218),CAAAGTATTTGGTCTCC (ASPH219), CAAAGTATTTGGTCTCC (ASPH220),CAAAGTATTTGGTCTCC (ASPH221), CAAAGTATTTGGTCTCC-TEG (ASPH222),CAAAGTATTTGGTCTCC-TEG (ASPH223), CAAAGTATTTGGTCTC (M1-ASPH47),CAAAGTATTTGGTCT (M2-ASPH47), CAAAGTATTTGGTC (M3-ASPH47),AAAGTATTTGGTCTCC (M4-ASPH47), AAAGTATTTGGTCTC (M5-ASPH47),AAAGTATTTGGTCT (M6-ASPH47), AAAGTATTTGGTC (M7-ASPH47), AAGTATTTGGTCTCC(M8-ASPH47), AAGTATTTGGTCTC (M9-ASPH47), AAGTATTTGGTCT (M10-ASPH47),AAGTATTTGGTC (M11-ASPH47), AGTATTTGGTCTCC (M12-ASPH47), AGTATTTGGTCTC(M13-ASPH47), AGTATTTGGTCT (M14-ASPH47), AGTATTTGGTC (M15-ASPH47), andCAAAGTATTTGGTCTCC (ASPH47).5. Pharmaceutical composition comprising the antisense oligonucleotideand optionally a pharmaceutically acceptable carrier according to anyone of embodiments 1 to 4.6. Antisense oligonucleotide according to any one of embodiments 1 to 4or pharmaceutical composition according to embodiment 5 for use in amethod of preventing and/or treating a malignant and/or benign tumor, animmunologic disease, fibrosis, or an ophthalmic disease.7. Antisense oligonucleotide or pharmaceutical composition for useaccording to embodiment 6, wherein the tumor is selected from the groupconsisting of solid tumors, blood born tumors, leukemias, tumormetastasis, hemangiomas, acoustic neurmas, neurofibromas, trachomas,pyogenic granulomas, psoriasis, astrocytoma, acoustic neuroma, blastoma,Ewing's tumor, craniopharyngloma, ependymoma, medulloblastoma, glioma,hemangloblastoma, Hodgkins-lymphoma, medullablastoma, leukaemia,mesothelioma, neuroblastoma, neurofibroma, non-Hodgkins lymphoma,pinealoma, retinoblastoma, sarcoma, seminoma, trachomas, Wilm's tumor,or is selected from the group of bile duct carcinoma, bladder carcinoma,brain tumor, breast cancer, bronchogenic carcinoma, carcinoma of thekidney, cervical cancer, choriocarcinoma, cystadenocarcinome, embryonalcarcinoma, epithelial carcinoma, esophageal cancer, cervical carcinoma,colon carcinoma, colorectal carcinoma, endometrial cancer, gallbladdercancer, gastric cancer, head cancer, liver carcinoma, lung carcinoma,medullary carcinoma, neck cancer, non-small-cell bronchogenic/lungcarcinoma, ovarian cancer, pancreas carcinoma, papillary carcinoma,papillary adenocarcinoma, prostate cancer, small intestine carcinoma,prostate carcinoma, rectal cancer, renal cell carcinoma, skin cancer,small-cell bronchogenic/lung carcinoma, squamous cell carcinoma,sebaceous gland carcinoma, testicular carcinoma, and uterine cancer.8. Antisense oligonucleotide or pharmaceutical composition for useaccording to embodiment 6, wherein the ophthalmic disease is selectedfrom the group consisting of glaucoma, posterior capsular opacification,dry eye, macular degeneration, e.g., age-related macular degeneration,diabetic macular endma, cataract, proliferative vitreoretinopathy,Marfan and Loeys-Dietz syndrome.

What is claimed is:
 1. An antisense oligonucleotide comprising: 10 to 18nucleotides hybridizing with the TGF-beta2 nucleic acid sequence of SEQID NO. 1, wherein one or more nucleotide(s) of the oligonucleotideis/are modified, wherein the modified nucleotide is at least one of anLNA, an ENA, polyalkylene oxide-, 2′-fluoro-, 2′-O-methoxy-, or a2′O-methyl-modified nucleotide.
 2. The antisense oligonucleotide ofclaim 1, wherein the modified nucleotide is located at the 5′- and/or3′-end of the oligonucleotide.
 3. The antisense oligonucleotide of claim1, wherein the oligonucleotide comprises a sequence selected from thegroup consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO.10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ IDNO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19 andSEQ ID NO.
 20. 4. The antisense oligonucleotide of claim 1, wherein theoligonucleotide is selected from the group consisting of AGTATTTGGTCTCC(ASPH190), AAGTATTTGGTCTC (ASPH191), AAGTATTTGGTCTCC (ASPH192),CAAAGTATTTGGTCT (ASPH193), AGTATTTGGTCTCC (ASPH194), AGTATTTGGTCTCC(ASPH195), AGTATTTGGTCTCC (ASPH196), AGTATTTGGTCTCC (ASPH197),AAGTATTTGGTCTC (ASPH198), AGTATTTGGTCTCCA (ASPH199), AGTATTTGGTCTCCA(ASPH200), AGTATTTGGTCTCCA (ASPH201), AGTATTTGGTCTCCA (ASPH202),AGTATTTGGTCTCCA (ASPH203), AGTATTTGGTCTCCA (ASPH204), AGTATTTGGTCTCCA(ASPH205), AAGTATTTGGTCTCC (ASPH206), AAGTATTTGGTCTCC (ASPH207),AAGTATTTGGTCTCC (ASPH208), AAGTATTTGGTCTCC (ASPH209), AAGTATTTGGTCTCC(ASPH210), AAGTATTTGGTCTCC (ASPH211), CAAAGTATTTGGTCTCC (ASPH212),CAAAGTATTTGGTCTCC (ASPH213), CAAAGTATTTGGTCTCC (ASPH214),CAAAGCTATTTGGTCTCC (ASPH215), CAAAGTATTTGGTCTCC (ASPH216),CAAAGTATTTGGTCTCC (ASPH217), CAAAGTATTTGGTCTCC (ASPH218),CAAAGTATTTGGTCTCC (ASPH219), CAAAGTATTTGGTCTCC (ASPH220),CAAAGTATTTGGTCTCC (ASPH221), CAAAGTATTTGGTCTCC-TEG (ASPH222),CAAAGTATTTGGTCTCC-TEG (ASPH223), CAAAGTATTTGGTCTC (M1-ASPH47),CAAAGTATTTGGTCT (M2-ASPH47), CAAAGTATTTGGTC (M3-ASPH47),AAAGTATTTGGTCTCC (M4-ASPH47), AAAGTATTTGGTCTC (M5-ASPH47),AAAGTATTTGGTCT (M6-ASPH47), AAAGTATTTGGTC (M7-ASPH47), AAGTATTTGGTCTCC(M8-ASPH47), AAGTATTTGGTCTC (M9-ASPH47), AAGTATTTGGTCT (M10-ASPH47),AAGTATTTGGTC (M11-ASPH47), AGTATTTGGTCTCC (M12-ASPH47), AGTATTTGGTCTC(M13-ASPH47), AGTATTTGGTCT (M14-ASPH47), AGTATTTGGTC (M15-ASPH47), andCAAAGTATTTGGTCTCC (ASPH47).
 5. A pharmaceutical composition comprisingthe antisense oligonucleotide of claim 1 and a pharmaceuticallyacceptable carrier.
 6. A method for inhibiting and/or treating at leastone of a malignant tumor, a benign tumor, an immunologic disease, afibrosis, or an ophthalmic disease, comprising: administering either anantisense oligonucleotide or a pharmaceutical composition; saidantisense oligonucleotide comprising: 10 to 18 nucleotides hybridizingwith the TGF-beta2 nucleic acid sequence of SEQ ID NO. 1, wherein one ormore nucleotide(s) of the oligonucleotide is/arc modified, wherein themodified nucleotide is at least one of an LNA, an ENA, polyalkyleneoxide-, 2′-fluoro-, 2′-O-methoxy-, or a 2′O-methyl-modified nucleotidesaid pharmaceutical composition comprising: an antisense oligonucleotidecomprising: 10 to 18 nucleotides hybridizing with the TGF-beta2 nucleicacid sequence of SEQ ID NO. 1, wherein one or more nucleotide(s) of theoligonucleotide is/are modified, wherein the modified nucleotide is atleast one or an LNA, an ENA, polyalkylene oxide-, 2′-fluoro-,2′-O-methoxy-, or a 2′O-methyl-modified nucleotide; and apharmaceutically acceptable carrier.
 7. The antisense oligonucleotide ofclaim 3, wherein the oligonucleotide is selected from the groupconsisting of AGTATTTGGTCTCC (ASPH190), AAGTATTTGGTCTC (ASPH191),AAGTATTTGGTCTCC (ASPH192), CAAAGTATTTGGTCT (ASPH193), AGTATTTGGTCTCC(ASPH194), AGTATTTGGTCTCC (ASPH195), AGTATTTGGTCTCC (ASPH196),AGTATTTGGTCTCC (ASPH197), AAGTATTTGGTCTC (ASPH198), AGTATTTGGTCTCCA(ASPH199), AGTATTTGGTCTCCA (ASPH200), AGTATTTGGTCTCCA (ASPH201),AGTATTTGGTCTCCA (ASPH202), AGTATTTGGTCTCCA (ASPH203), AGTATTTGGTCTCCA(ASPH204), AGTATTTGGTCTCCA (ASPH205), AAGTATTTGGTCTCC (ASPH206),AAGTATTTGGTCTCC (ASPH207), AAGTATTTGGTCTCC (ASPH208), AAGTATTTGGTCTCC(ASPH209), AAGTATTTGGTCTCC (ASPH210), AAGTATTTGGTCTCC (ASPH211),CAAAGTATTTGGTCTCC (ASPH212), CAAAGTATTTGGTCTCC (ASPH213),CAAAGTATTTGGTCTCC (ASPH214), CAAAGCTATTTGGTCTCC (ASPH215),CAAAGTATTTGGTCTCC (ASPH216), CAAAGTATTTGGTCTCC (ASPH217),CAAAGTATTTGGTCTCC (ASPH218), CAAAGTATTTGGTCTCC (ASPH219),CAAAGTATTTGGTCTCC (ASPH220), CAAAGTATTTGGTCTCC (ASPH221),CAAAGTATTTGGTCTCC-TEG (ASPH222), CAAAGTATTTGGTCTCC-TEG (ASPH223),CAAAGTATTTGGTCTC (M1-ASPH47), CAAAGTATTTGGTCT (M2-ASPH47),CAAAGTATTTGGTC (M3-ASPH47), AAAGTATTTGGTCTCC (M4-ASPH47),AAAGTATTTGGTCTC (M5-ASPH47), AAAGTATTTGGTCT (M6-ASPH47), AAAGTATTTGGTC(M7-ASPH47), AAGTATTTGGTCTCC (M8-ASPH47), AAGTATTTGGTCTC (M9-ASPH47),AAGTATTTGGTCT (M10-ASPH47), AAGTATTTGGTC (M11-ASPH47), AGTATTTGGTCTCC(M12-ASPH47), AGTATTTGGTCTC (M13-ASPH47), AGTATTTGGTCT (M14-ASPH47),AGTATTTGGTC (M15-ASPH47), and CAAAGTATTTGGTCTCC (ASPH47).